Loss of Heterozygosity (LOH) for HumACTBP2 (SE33) Is Detected in Philadelphia (Ph)-Positive and Ph-Negative Hematopoietic Cells of Some Patients with Ph-Positive CML.

Introduction: In the majority of patients with Philadelphia (Ph)-positive chronic myelogenous leukemia (CML), the non-leukemic hematopoiesis is considered polyclonal. Occasionally, clonal cytogenetic abnormalities have been reported in Ph-negative cells from patients in cytogenetic remission after IFN-alpha or imatinib. In the present project we determined clonality of flow-sorted hematopoietic cells from untreated and treated patients with Ph-positive CML in first chronic phase by analyzing the two alleles of short tandem repeats (STR) of given loci. The loss of one of the two alleles of STR identifies loss of heterogzygosity (LOH) and clonal expansion. These results were compared to conventional cytogenetics and correlated with response to therapy.

Patients and methods: A total of 25 consecutive patients (13 m/12 f, median age 58 y) treated at the University of Leipzig from April to June 2005 were included. Conventional cytogenetics were analyzed by R-banding. FISH using the LSI BCR/ABL ES probe in unsorted as well as in flow-sorted monocytes (CD14+), granulocytes (CD14-/CD33+), T- lymphocytes (CD3+), B-lymphocytes (CD19+), and CD34+-cells was performed. For LOH analysis, DNA was extracted from flow-sorted cells. 8 autosomal loci were analysed: D3S1358 (3p), vWA (12p12-pter), D8S1179 (8q), D21S11 (21q11.2-q21), D18S51 (18q21.3), TH01 (11p15.5), FGA (4q28), SE33 (6q15). PCR products were detected by capillary electrophoresis. Loss of, or a strong decrease in the signal intensity of one allele was considered as evidence for LOH. An allelic imbalance factor (AIF) was calculated by the quotient of the peak ratios from constitutional and flow-sorted cell DNA (N1:N2/T1:T2). An AIF > 1.8 was considered as LOH. Buccal swabs were used as controls.

Results: Median time from diagnosis to analysis was 60 months. Of the 25 patients, twenty (80%) received Imatinib for a median of 26 months, three (12%) were newly diagnosed and two (8%) were treated with IFN-alpha. Both patients with IFN-alpha as well as 8 (40%) patients treated with Imatinib had sustained complete cytogenetic remission (CCR). Furthermore, 8 (40%) responded to Imatinib with hematological remission (HR) only, 3 (15%) patients with major CR (MCR) and one patient (5%) with minor CR. Conventional cytogenetics revealed additional abnormalities [t(7;17), del (20q), trisomy 8, and -Y] in Ph- negative cells in 4 (16%) patients. No LOH was detected in these patients. Ph-positive B-cells were detected in 4 (16%) with HR. Interestingly, the median quotient of Ph-positive CD34+ and Ph-positive unsorted cells was 0.65. LOH for SE33 in CD34+ cells, monocytes and granulocytes was detected in 6 (24%) patients [3 in CCR (2 patients treated with IFN-alpha and 1 with imatinib)], 1 patient in MCR under imatinib, and 2 patients with newly diagnosed CML. LOH was not detected in buccal swabs.

Conclusions: We confirm the occurrence of clonal cytogenetic abnormalities in Ph-negative cells. LOH did not parallel abnormal cytogenetics and LOH for SE33 only was detected in patients with Ph-negative and Ph-positive cytogenetics. The detection of LOH which could be a sign of inactivation of tumor suppressor genes in treated and untreated patients with CML argues against a treatment-induced event and raises speculations on the role of this finding in the pathogenesis of at least some cases of CML.